The present invention relates to providing a SPR sensor array capable of simultaneously sensing a plurality of samples, to methods for its production, measurement assemblies as well as to adjustment and measurement methods for parallel readout of the sensor system and to use thereof in the search for active substances and in high-throughput screening.
One modern approach in the search for active substances involves generating a large number of diverse chemical compounds by means of automated synthesizers, this plurality of diverse structures then being tested for binding to interaction partners often represented by biomacromolecules such as proteins. One automated method which assays a large number of samples in this way is also termed high-throughput screening.
Due to the biological dispersion of the results in studying the bindings it is particularly important to achieve exactly the same conditions for all compounds in the binding test. This is why the test ideally should be implemented for all samples simultaneously, where possible, and with the same solution of the interaction partner under test to eliminate the effects of ageing and temperature drift as well as differences in the binding times for the compounds. Due to complexities involved in purifying biomacromolecules the quantities needed for the test should be kept to a minimum.
One particularly effective method of implementing the binding test is surface plasmon resonance (SPR) spectroscopy. Unlike fluorescence and chemiluminescence methods no dye-marked samples and also no antibodies are needed in SPR for the protein to be tested. In SPR an interaction partner (e.g. ligand) is immobilized on a metal surface and its binding to another interaction partner (e.g. receptor) demonstrated. For this purpose an optical substrate (usually a prism) is coated with gold and the drop in internal reflectivity in the prism detected as a function of the set angle or as a function of the wavelength (Kretschmann configuration). What is demonstrated ultimately is a change in the refractive index of the medium at the side opposite the gold film which occurs when molecules bind to the surface.
FIG. 1a shows diagrammatically the so-called Kretschmann geometry which is often used to measure the SPR effect. In this case a thin gold film 125 applied to a prism 20 is brought into wetting contact with the solution 160 to be assayed. What is usually measured is the internal reflectivity at glass/gold/fluid interfaces either as a function of the angle of incidence ∂ or as a function of the wavelength xcex. At a suitable resonance condition the reflectivity is greatly reduced, the energy of the light then being converted into electron charge density waves (plasmons) along the gold/fluid interface. The condition for resonance approximates (from Chapter 4, xe2x80x9cSurface Plasmon Resonancexe2x80x9d in G. Ramsay, Commercial Biosensors, John Wiley and Sons (1998) ) to:                     2        ⁢        π            λ        ⁢          n      prism        ⁢    sin    ⁢          xe2x80x83        ⁢    ϑ    ≈                    2        ⁢                  xe2x80x83                ⁢        π            λ        ⁢                                                      n              metal              2                        ⁡                          (              λ              )                                ⁢                      n            sample            2                                                              n              metal              2                        ⁡                          (              λ              )                                +                      n            sample            2                              
where nprism is the refractive index of the prism, nmetal the complex refractive index of the metal coating and nsample that of the sample. ∂ and xcex stand for the angle of incidence and wavelength of the incident light, respectively. The wavelength spectra (FIG. 1b) respectively the angle spectra (FIG. 1c) exhibit a reduction in reflectivity in the wavelength range or in the angle range respectively in which the resonance condition as cited above is satisfied. Changing the refractive index in the solution nsample alters the resonance condition, as a result of which the resonance curves are shifted by a value which for small changes in the refractive index is linear to this change (a calibration being made, where necessary, for larger changes). Since the reflected light penetrates into the fluid only by a few 100 nm the change in the refractive index is measured locally in this range. When the target molecules (e.g. proteins) 162 in the solution bind to suitable interaction partners 161 immobilized on the surface (i.e. association and dissociation forming an equilibrium) the local concentration of the target molecule at the surface increases which can then be demonstrated as a change in the refractive index.
WO 99/60382 describes an SPR sensor capable of simultaneously sensing a plurality of samples. A measurement assembly for reading out such a SPR sensor system in parallel is disclosed in WO 00/31515. This proposes an apparatus for implementing SPR measurements on a plurality of samples in parallel which is based on the principle of wavelength measurement, but does not use a prism, but an array of sensor fingers capable of carrying another substance on each sensor finger. This array can be coated in a microtiter plate (MTP) and measured, i.e. each sensor finger is able to measure another solution. The contrast between the sensor fields and the intermediate regions is dictated by the geometry of the waveguides. In this case light passes through the array only at the regions at which a sensor field is applied, resulting in a high contrast. The disadvantage is the expense in producing the sensor fingers and their sensitiveness to physical contact as well as the relatively high sample consumption in coating.
WO 98/34098 shows sample fields on an SPR-compatible gold film applied to a prism. The contrast is determined by setting suitable resonance conditions. The disadvantage here is that the surfaces need to be very homogenous since it is only the region of the sensor surface area that shows a contrast in imaging under SPR conditions that exhibits the same layer thicknesses.
Another SPR imaging system is described in B. P. Nelson et al., Anal. Chemical 1999, 71, pages 3928-3934. In this case a uniform gold surface applied to a non-structured glass plate is patterned with an array of 500xc3x97500 xcexcm large squares covered with DNA, the DNA squares being separated by squares covered with alkanethiol intended to prevent adsorption of the protein outside of the DNA squares. The DNA squares are then brought into contact with a protein sample and an image of the gold surface produced at the SPR angle on an CCD chip before and after contact is made. Here, distinguishing the DNA squares from the other regions depends on the molecular weight of the immobilized chemical or biological molecules, the contrast sinking with a reduction in the molecular weight. Also a disadvantage in this system is the relatively large pixel region to which a DNA square needs to be assigned on the CCD camera to ensure adequate contrast. These requirements conflict with the need for a miniaturized SPR sensor array for universal application.
Described in WO 90/05305 is a replaceable sensor unit for use in an optical biosensor system (WO 90/05295) in which the geometry and arrangement of the sample fields on the non-structured sensor unit is not dictated by the latter. Assigning the sample fields on the sensor unit is done by bringing it into contact with a block unit for handling the fluids, e.g. the throughflow system as disclosed in WO 90/05295, the throughflow system defining the arrangement of the sensor surfaces one-dimensionally (one-dimensional array). The disadvantage in this case is that making use of a throughflow system makes it difficult to use and miniaturize a two-dimensional sample array (two-dimensional array).
The present invention is based on the object of providing an improved SPR sensor array.
This object is achieved by the characterizing features of the claim 1 and the subject matter of parallel claims respectively. Advantageous aspects are the subject matter of the dependent claims.
In accordance with the invention separating means or separators are provided for structuring the SPR sensor array so that a two-dimensional sample array is made possible. A plurality of samples is arranged in a two-dimensional sensor array such that the geometry and number of the sensor fields or sensor surface areas as well as the contrast between sensor fields and their intermediate regions are determined by separating means on the sensor system and the surface areas of the sensor fields are located parallel to the coordinate plane of the sample array. Since the separating means create a contrast outside of surface plasmon resonance occurring in the SPR sensor surface areas, positioning and adjusting a sensor array in a measurement assembly can now be done directly in that practically any radiation can be directed to the array, permitting a setting due to the contrast produced between the SPR sensor surface areas and the separating means, since this enables these regions to be easily distinguished from each other in imaging or the individual SPR sensor surface areas to be easily distinguished from each other.
In other words, whereas in prior art as per B. P. Nelson et al. (see above) the variable physical conditions (e.g. the angle of incidence of the radiation on the sensor array or the wavelength of the radiation) needs to be regulated highly precisely to the resonance to permit distinguishing the regions to be analyzed from each other in imaging, since in this case outside of the resonance similar reflection occurred from the regions spotted with DNS and with alkanethiol on the gold, now in the present invention sensing can be done with a radiation under practically any physical condition (any angle or any wavelength) and the existing contrast permits distinguishing the regions. The molecular weight of the chemical compound to be immobilized can also be selected optionally, thus also permitting the application of small organic molecules (smaller than 5000, preferably smaller than 1000, even better smaller than 500 Dalton).
Although it is possible that the reflection in the region of the separating means outside of resonance occurring in the SPR sensor surface areas is smaller than in the region of the SPR sensor surface areas, the SPR sensor surface areas and the separating means are configured so that the reflectivity of the separating means is less than the reflectivity the SPR sensor surface areas, i.e. at least outside of resonance occurring in the SPR sensor surface areas. The absorption in the resonance range is possibly so strong that the reflectivity in these SPR sensor surface areas at resonance is less than the reflectivity of the separating means. This merely results in an inversion in the contrast in the resonance range so that distinguishing the regions in imaging continues to be possible directly. The reflection spectrum of the SPR sensor surface areas intersects the (preferably constant) reflectivity of the separating means, at two points so that it is only precisely at these points that no contrast occurs which is obviously negligible and doubtlessly a major advantageous over the system as per B. P. Nelson et al. It is, however, preferred in the present invention to configure the separating means and SPR sensor surface areas so that the reflectivity of the SPR sensor surface areas is always greater than the reflectivity of the separating means, also in the resonance range in the SPR sensor surface areas.
In one preferred embodiment the separating means are directly applied to the sensor system. Achieving the separating means and the sensor surface areas can be performed in suitable ways, it is thus being possible to apply a radiation-absorbing substance to the sensor substrate as the separating means whilst an SPR-compatible material, e.g. a metal, preferably gold, is deposited as the SPR sensor surface area. It is possible to make use of a material as the separating means which has a refractive index equal to a larger than (preferably larger by max. 0.1) than that of the substrate material so that the radiation although refracted from the substrate into the separating means does not permit refraction back into the substrate. This can also be combined with the use of an absorbing material, by introducing namely into the separating means with the higher refractive index additional radiation-absorbing substances such as e.g. carbon or a dye. Preferably the thickness and width of the layer comprising the contrast-forming material is determined so that radiation refracted from the substrate into the layer is reflected back to the substrate maximally twice at the side of the layer facing away from the substrate surface.
Suitable materials for the separating means are absorbing layers of metal or a semiconductor or polymers (e.g. photoresist, silicon).
Preferably the separating means assure in addition that no contamination between the sensor fields or SPR sensor surface areas can occur. This is achieved by the separating means forming elevations above the SPR sensor surface areas in the direction perpendicular to the substrate preferably 0.01 mm to 5 mm differing in height. It is good practice when the flanks or surfaces of the separating means forming vessels for receiving a sample fluid are hydrophobic or hydrophobicized so that an aqueous solution is well contained without the possibility of cross-contamination with other SPR sensor surface areas.
In one aspect the SPR sensor array consists of a prism coated with a SPR-compatible layer of metal, where necessary, provided with an adhesive-promoting film as well as with the separating means.
In another aspect the SPR sensor array multiply consists of a sample-carrying sensor array provided with separating means and SPR sensor surface areas and a beam-guiding component. The beam-guiding component consists preferably of a prism. In addition an optical mediator for suitably adapting the refractive index may be provided between the beam-guiding component and the sample-carrying sensor unit.
Minor inhomogenities in the thickness of the gold film (up to 2-3 nm) are acceptable in these arrays since the image of the sensor surface area is visible irrespective of SPR resonance.
In addition the invention relates to a measurement assembly containing the SPR sensor array for parallel measurement of a plurality of preferably differing samples which can be produced at low cost in many copies and thus also suitable for once-only use to thus reduce substance consumption for coating a sensor field as compared to prior art sensor systems.
For examining a plurality of differing samples for interaction by the SPR method it is good practice to arrange them on a substrate two-dimensionally (two-dimensional array) and to image them in parallel e.g. with the aid of a CCD camera. In analyzing the image recorded by such a spatial resolution detector it is an enormous advantageous that the invention produces a strong light/dark contrast between the regions spotted by the samples (sensor fields) and the intermediate regions to achieve a sharp image of the sensor fields which permits an improved assignment of the physical spatial coordinates of the samples on the substrate to the coordinates in the image.
For this purpose prior art made use of the SPR effect itself, the contrast being produced solely by setting suitable resonance conditions e.g. by setting a suitable angle for an angle-dependent measurement. Since in resonance too, the light is not totally converted into surface plasmons this method of producing the contrast is at a disadvantage as compared to the present invention (10-20% of the light is also reflected in resonance), this likewise making high demands on the homogeneity of the thickness of the gold film. Better results are obtainable by employing structured, absorbing layers, i.e. the separating means of the present invention.
Common to all approaches of the prior art as cited for parallel sensing a plurality of samples is that the contrast between the fields spotted by the samples and the intermediate regions is not dictated by contrast-generating separating means as taught by the present invention.